GB 4789.42-2016 PDF in English
GB 4789.42-2016 (GB4789.42-2016) PDF English
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Detection of norovirus in shellfish -- Conventional RT-PCR and real-time RT-PCR
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Standards related to: GB 4789.42-2016
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GB 4789.42-2016: PDF in English GB 4789.42-2016
GB
NATIONAL STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Food microbiological examination –
Examination of norovirus
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
3. No action is required - Full-copy of this standard will be automatically &
immediately delivered to your EMAIL address in 0~60 minutes.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Reagent ... 5
4 Testing procedures ... 6
5 Operating procedures ... 6
6 Results and reports ... 12
Appendix A Real-time fluorescent RT-PCR primers and probes ... 14
Appendix B Real-time fluorescent RT-PCR reaction system and parameters 15
Appendix C Process control virus culture and primers, probes ... 16
Appendix D Preparation of externally added amplification control RNA ) ... 18
Appendix E RNase removal and RNase-free solution preparation ... 20
Foreword
This standard replaces SN/T 1635-2005 “Detection of norovirus in shellfish -
Conventional RT-PCR and real-time RT-PCR”.
As compared with SN/T 1635-2005, the main changes of this standard are as
follows.
- CHANGE the standard name changed into “National food safety standard
- Food microbiological examination - Examination of norovirus”;
- EXTEND the standard test range from “shellfish” to “food”;
- MODIFY the “operational procedures”;
- ADD the “quality control requirements”, as shown in Appendix C;
- DELETE the “normal RT-PCR method”.
National food safety standard -
Food microbiological examination –
Examination of norovirus
1 Scope
This standard specifies real-time fluorescent RT-PCR detection method of
norovirus in food.
This standard applies to the norovirus nucleic acid detection in hard-surfaced
foods such as shellfish, raw vegetables, carrots, melons, nuts and so on AND
such soft foods as the strawberries, tomatoes, grapes and so on.
2 Equipment and materials
In addition to routine sterilization and culture equipment for microbiological
laboratories, other equipment and materials are as follows.
2.1 Real-time fluorescence PCR instrument.
2.2 Freeze centrifuge.
2.3 Sterile blade or equivalent homogenizer.
2.4 Scrollers.
2.5 Balance. sensitivity of 0.1 g.
2.6 Oscillator.
2.7 Water bath.
2.8 Centrifuge.
2.9 Autoclave pot.
2.10 Low temperature refrigerator. -80 °C.
2.11 Micro-pipettes.
2.12 pH meter or precision pH test paper.
5.1.1.2 MOVE the sample piece into a sample bag with a 400 mL mesh filter
bag; ADD 40 mL of TGBE solution (for soft fruit sample, it is needed to add 30
U of A.niger pectinase or 1140 U of A.aculeatus pectinase); ADD 10 μL of
process control virus.
5.1.1.3 At room temperature, MAKE it subject to oscillation for 20 min at the
speed of 60 times/min. As for the acid soft fruit, it shall detect the pH value at
the interval of 10 min during oscillation; if the pH is less than 9.0, USE the 1
mol/L NaOH to adjust the pH to 9.5; EXTEND the oscillation duration for 10
min every time the pH is adjusted.
5.1.1.4 TRASNFER the oscillated solution into the centrifuge tube. If the
volume is large, it may use two centrifuge tubes. CONTRIFUGE it at 4 °C for
30 min at the speed of 10000 r/min. TAKE the supernatant; PLACE it into a
clean test tube or conical flask; USE 1 mol/L HCl to adjust the pH to 7.0.
5.1.1.5 ADD 0.25 times volumes of 5 x PEG/NaCl solution so that the final
solution concentration is 100 g/L PEG and 0.3 mol/L NaCl. SHAKE it uniformly
within 60 s; MAKE it subject to oscillation at 4 °C for 60 min at the speed of 60
times/min. CENTRIFUGE it at 4 °C for 30 min at the speed of 10000 r/min;
DISCARD the supernatant; CENTRIFUGE it at 4 °C for 5 min at the speed of
10000 r/min to solidify the precipitation; DISCARD the supernatant.
5.1.1.6 500 μL of PBS suspension precipitation. If the food sample is a raw
vegetable, the suspension can be transferred directly to a clean test tube, and
the millimeters of suspension is determined and recorded, for subsequent RNA
extraction. If the food sample is soft fruit, the suspension is transferred into a
chloroform-resistant test tube. ADD 500 μL of chloroform/butanol mixed
solution; MIX it uniformly in vortex; LET it stand at room temperature for 5 min.
CENTRIFUGE it at 4 °C for 15 min at the speed of 10000 r/min; carefully
TRANSFER the liquid phase part into a clean test tube; DETERMINE the
RECORD the millimeters of suspension, for the subsequent RNA extraction.
5.1.2 Hard surfaced food
5.1.2.1 USE PBS to wet the sterile cotton swab; WIPE the food surface (< 100
cm2) with force; RECORD the wipe area. ADD 10 μL of the process control
virus to this swab.
5.1.2.2 IMMERSE the cotton swab in a 490 μL PBS test tube; PRESS it onto
the tube wall to extrude the solution out. REPEAT the immersion and extrusion
for 3 ~ 4 times, to ensure extruding out the maximum amount of virus;
DETERMINE and RECORD the millimeters of solution, for the subsequent
RNA extraction. Since the hard-surfaced food surface may be too rough AND it
may damage the cotton swab, so it may use several cotton swabs.
ADD the same volume of isopropyl alcohol into the centrifuge tube; MAKE it
upside down to mix it uniformly; PLACE it at room temperature for 5 min;
CENTRIFUGE it for 5 min at the speed of 12000 r/min; DISCARD the
supernatant; PLACE it upside down onto the absorbent paper to make it dry
(different samples must be dried at different positions of the absorbent paper).
5.2.3 Virus RNA purification
5.2.3.1 ADD the equal volume of 75% ethanol each time; MAKE it upside down
to rinse the RNA precipitation twice.
5.2.3.2 CENTRIFUGE it at 4 °C for 10 min at the speed of 12000 r/min;
carefully DISCARD the supernatant; PLACE it upside down on the absorbent
paper to make it dry (different samples must be dried at different positions of
the absorbent paper). Carefully POUR off the supernatant; USE the
micro-sampler to make it dry; USE one absorption tip for one sample; DO not
let the absorption tip touch the precipitation; DRY it at room temperature for 3
min; AND it shall not be over-dried to avoid RNA insoluble.
5.2.3.3 ADD 16 μL of RNase-free ultrapure water; MIX it uniformly and gently;
DISSOLVE the RNA on the tube wall; CENTRIFUGE it for 5 s at the speed of
2000 r/min; PRESERVE it on ice to prepare for use.
5.3 Quality control
5.3.1 Blank control
USE the RNase-free ultrapure water as a blank control (A reaction hole).
5.3.2 Negative control
EXTRACT the RNA from the shellfish containing no norovirus as a negative
control (B reaction hole).
5.3.3 Positive control
USE the externally added amplification control RNA as a positive control (J
reaction hole).
5.3.4 Process control viruses
5.3.4.1 USE the extraction efficiency of the process control virus RNA in the
food to indicate the extraction efficiency of the norovirus RNA in the food, as
the virus extraction process control.
5.3.4.2 EXTRACT and PURIFY the RNA of the process control virus in
accordance with the procedures in 5.2. It may extract it in large amount;
added amplification control RNA) Ct value, that is, the inhibition index = (H
reaction hole) Ct value - (J reaction hole) Ct value. If the inhibition index is ≥
2.00, it shall compare the inhibition index of the 10-fold diluted food sample,
that is, the inhibition index = (I reaction hole) Ct value - (J reaction hole) Ct
value.
5.4 Real-time fluorescent RT-PCR
Real-time RT-PCR reaction system and reaction parameters are described in
Appendix B. The amount of each reagent in the reaction system can be
appropriately adjusted depending on the particular situation or the different
total reaction volume. A commercially available real-time fluorescent RT-PCR
kit can be used. It can also increase and adjust the reaction hole to realize the
independent detection of the GI type and GII type norovirus in one time. After
adding the 18.5 μL of real-time fluorescent RT-PCR reaction system into the
reaction hole, ADD the following substances into different reaction holes to
detect the GI or GII genotype norovirus.
A reaction hole. blank control, ADD 5 µL of RNase-free ultrapure water + 1.5
µL of GI or GII type primer probe;
B reaction hole. negative control, ADD 5 µL of negative extraction control RNA
+ 1.5 µL of GI or GII type primer probe;
C reaction hole. virus extraction process control 1, ADD 5 μL of process control
virus contained food sample RNA + 1.5 μL of process control virus primer
probe;
D reaction hole. virus extraction process control 2, ADD 5 μL of process control
virus RNA + 1.5 μL of process control virus primer probe;
E reaction hole. virus extraction process control 3, ADD 5 μL of 10-1 fold
dilution process control virus RNA + 1.5 μL of process control virus primer
probe;
F reaction hole. virus extraction process control 4, ADD 5μL of 10-2 fold dilution
process control virus R...
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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